Plastid phylogenomics and fossil evidence provide new insights into the evolutionary complexity of the ‘woody clade’ in Saxifragales

Background The “woody clade” in Saxifragales (WCS), encompassing four woody families (Altingiaceae, Cercidiphyllaceae, Daphniphyllaceae, and Hamamelidaceae), is a phylogenetically recalcitrant node in the angiosperm tree of life, as the interfamilial relationships of the WCS remain contentious. Based on a comprehensive sampling of WCS genera, this study aims to recover a robust maternal backbone phylogeny of the WCS by analyzing plastid genome (plastome) sequence data using Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP) methods, and to explore the possible causes of the phylogenetic recalcitrance with respect to deep relationships within the WCS, in combination with molecular and fossil evidence. Results Although the four WCS families were identically resolved as monophyletic, the MP analysis recovered different tree topologies for the relationships among Altingiaceae, Cercidiphyllaceae, and Daphniphyllaceae from the ML and BI phylogenies. The fossil-calibrated plastome phylogeny showed that the WCS underwent a rapid divergence of crown groups in the early Cretaceous (between 104.79 and 100.23 Ma), leading to the origin of the stem lineage ancestors of Altingiaceae, Cercidiphyllaceae, Daphniphyllaceae, and Hamamelidaceae within a very short time span (∼4.56 Ma). Compared with the tree topology recovered in a previous study based on nuclear genome data, cytonuclear discordance regarding the interfamilial relationships of the WCS was detected. Conclusions Molecular and fossil evidence imply that the early divergence of the WCS might have experienced radiative diversification of crown groups, extensive extinctions at the genus and species levels around the Cretaceous/Paleocene boundary, and ancient hybridization. Such evolutionarily complex events may introduce biases in topological estimations within the WCS due to incomplete lineage sorting, cytonuclear discordance, and long-branch attraction, potentially impacting the accurate reconstruction of deep relationships. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-024-04917-9.

Given that fossils represent the remains of past plants, they have the capacity to unveil intermediate evolutionary connections from the geological past, thereby offering valuable evidence for comprehending the evolution of the tree of life [34][35][36][37].The combination of molecular and fossil evidence is recommended as an efficient approach for inferring the evolutionarily complex events that might result in phylogenetic recalcitrance in the angiosperm tree of life [16,30,38,39].Recently, genome-scale sequence data, particularly plastid genomes (plastomes), have been increasingly used to solve historical puzzles in plant phylogenetics [e.g, 8,28,29,[40][41][42][43][44][45][46][47][48].In general, phylogeny inferred from uniparentally inherited plastomes reflects only the maternal (or, in some cases, paternal) relationships, as compared to the more comprehensive evolutionary history recovered through the phylogenetic analyses of biparentally inherited nuclear genomes [49].Nevertheless, the comparison of tree topologies inferred from plastomes and nuclear genomes can facilitate the determination of phylogenetic discordance between plastid and nuclear datasets (cytonuclear discordance), providing robust evidence to infer whether the evolution of the taxa in question has involved evolutionarily complex events, such as incomplete lineage sorting and hybridization [50,51].
Recent phylogenomic studies have yielded valuable insights into the deep relationships within the WCS.Based on nuclear genome data generated by target-capture sequencing and RNA-seq, Folk et al. [28] recovered the phylogenetic backbone of the WCS with high-resolution and well-supported interfamilial relationships.In contrast, previous plastome-based phylogenetic analyses of the WCS taxa have demonstrated tree topologies with low support [52,53] and inconsistent inter-familial relationships [32,33].Consequently, the family-level maternal relationships within the WCS remain ambiguous, providing weak evidence for detecting cytonuclear discordance in the deep relationships within the WCS.Notably, the maternal inheritance of plastomes in the WCS as well as in the Saxifragales has been verified by the investigations of several species including Hamamelis virginiana, Heuchera micrantha, and Tolmiea menziesii [54,55].By expanding the taxonomic sampling of the WCS to include the majority of genera, the objectives Table 1 Comparison of the order-level taxonomic placements of the four families within the "woody clade" in Saxifragales among previous morphology-based classification systems of this study are to recover a robust maternal backbone phylogeny of the WCS through analysis of plastome sequence data.Based on a time-calibrated phylogenetic framework, fossil evidence, and a comparison of plastome (this study) and nuclear [28] phylogenies, evolutionarily complex events putatively responsible for the phylogenetic recalcitrance of the deep phylogeny of the WCS were inferred.

Plastome features
The sampled WCS plastomes showed a typical quadripartite structure, encompassing a large single-copy (LSC) region and a small single-copy (SSC) region separated by two inverted repeat (IRa and IRb) regions (Fig. S1; Tables S1, S2 and S3).The genome sizes ranged from 158,149 bp to 160,861 bp, and GC content varied from 37.7 to 38.2% (Table S3).The WCS plastomes were highly conserved in terms of gene content and structure (Fig. S2; Tables S3  and S4), and they possessed 115 unique genes, including 81 protein-coding genes (PCGs), 30 transfer RNAs (tRNAs), and four ribosomal RNAs (rRNAs).Except for the pseudogenization of ycf15 in Chunia bucklandioides, none gene deletion was found in the WCS plastomes (Fig. S2).

Phylogenetic relationship
ML and BI analyses yielded highly congruent tree topologies (Figs.

Estimation of divergence time
Fossil-calibrated molecular dating (Fig. 3) showed that the diversification of the WCS crown groups initiated at ∼104.

Discussion
The fossil-calibrated plastome phylogeny (Fig. 3) showed that all three of the WCS monotypic families (i.e., Altingiaceae, Cercidiphyllaceae, and Daphniphyllaceae) are   [59,62].Within Cercidiphyllaceae, four extinct genera (Jenkinsella, Joffrea, Nyssidium, and Trochodendroides) have been reported [63][64][65][66].Among them, a Joffrea species has been documented from the late Paleocene of Canada [64]; 10 Jenkinsella, 13 Nyssidium, and 60 Trochodendroides species [67,68] have been discovered across the Northern Hemisphere spanning from early Cretaceous Fig. 3 Fossil-calibrated molecular phylogenetic dating of the "woody clade" in Saxifragales.The chronological estimates were performed using MCMCtree with the maximum likelihood (ML) tree as a topological constraint.Numbers superimposed on the branches represent mean divergent ages and 95% confidence interval of each node, respectively.The red dots represent the secondary calibration and fossil-calibration nodes.The divergence time and timeline are indicated in million years ago (Ma).FO, Fothergilleae; HA, Hamamelideae; EU, Eustigmateae; CO, Corylopsideae; LO, Loropetaleae to Eocene epochs [67,68].Most of these extinct taxa have been identified through well-preserved reproductive organs utilizing various approaches such as scanning electron microscopy and cladistic analysis to explore their taxonomic affinities.The aforementioned evidence strongly supports that cladogenesis did occur during the early evolution of WCS clades.Although no Daphniphyllaceae fossil has yet been found, the currently available fossil evidence robustly supports the conclusion that the deep stems of the afore-mentioned WCS clades can be attributed to the prominent extinction of their closely related taxa during their initial evolutionary stages.
For phylogenetic analysis, the families Altingiaceae, Cercidiphyllaceae, and Daphniphyllaceae exhibit extremely deep stems that may expose them to longbranch attraction [73], potentially leading to erroneous grouping in evolutionary trees.Previous studies have demonstrated that MP phylogenies are more susceptible to long-branch attraction compared to BI and ML phylogenies [74,75].Consistent with this, our MP phylogeny revealed distinct tree topologies concerning the interfamilial relationships among Altingiaceae, Cercidiphyllaceae, and Daphniphyllaceae when compared to ML and BI phylogenies (Figs. 1 and 2).These findings suggest that the reconstruction of the WCS's phylogeny could be influenced by long-branch attraction effects, resulting in a certain degree of bias in topological estimation [73,75,76].Consequently, significant extinction events during the early evolution of the WCS might contribute significantly to the recalcitrance observed in resolving deep relationships within this clade.
Previous studies have revealed that both BI and ML phylogenies are more resistant to long-branch attraction than MP phylogenies [73][74][75].The following discussion is mainly based on the BI and ML tree topologies because the interfamilial relationships recovered from the BI and ML phylogenies can be more reliable than those recovered from the MP phylogenies.In this study, both BI and ML phylogenies recovered three successively divergent clades within the WCS, corresponding to Altingiaceae, Cercidiphyllaceae + Daphniphyllaceae, and Hamamelidaceae.The interfamilial relationships are consistent with those revealed by Jian et al. [21], Soltis et al. [27], Xiang et al. [30], Tarullo et al. [31], and Bi et al. [33].Nevertheless, the interfamilial relationships recovered in this study are inconsistent with those obtained by analyzing plastid sequence data alone, which proposed topologies of (Daphniphyllaceae, (Altingiaceae, (Hamamelidaceae, Cercidiphyllaceae))) [32,77], (Altingiaceae, (Cercidiphyllaceae, (Daphniphyllaceae, Hamamelidaceae))) based on 83 plastid PCGs of four species [52], (Hamamelidaceae, (Cercidiphyllaceae, Daphniphyllaceae, Altingiaceae)) based on 83 protein-coding genes, and ((Daphniphyllaceae, Altingiaceae), (Hamamelidaceae, Cercidiphyllaceae)) based on plastome of nine species [53].Due to the limited taxonomic sampling of the WCS, these studies likely suffered from phylogenetic errors.Based on a more comprehensive sampling of the WCS taxa at the genus level and the concatenated 78 plastid PCGs that contains more sequence variations and phylogenetically informative sites than was available in previous plastid phylogenetic studies, the deep relationships within WCS recovered in this study were robustly supported.This led to the recovery of the maternal backbone phylogeny of the WCS, providing new insights for inferring the evolutionarily complex events that likely caused the phylogenetic recalcitrance of the deep relationships within WCS.
Topologically, the deep relationships of the WCS recovered in this study are incongruent with those recovered based on the phylogenomic analysis of targetcapture sequencing and transcriptome data [28], which proposed a successive divergence of Daphniphyllaceae, Cercidiphyllaceae, Altingiaceae, and Hamamelidaceae with robust support for each node.Based on these results, phylogenetic incongruence between plastid and nuclear data (cytonuclear discordance) was detected in the deep clades of the WCS (Fig. 4).Cytonuclear discordance is commonly observed in some phylogenetically recalcitrant plant lineages [16,50,[78][79][80][81]; in most cases, nuclear phylogeny is more congruent with morphologic characteristics than plastid phylogeny, and such discordance is thought to be caused by hybridization [17,50,82,83].
By comparing the plastome (this study) and nuclear genome phylogenies [28] with the morphological characteristics, we found that the interfamilial relationships recovered based on the analyses of target-capture sequencing and transcriptome data [28] are more consistent with the morphologies.Specifically, Altingiaceae has traditionally been treated as a member of Hamamelidaceae [10,11,13].Both families are monoecious and have one or two-chambered anthers, distinct from the dioecism and four-chambered anthers of Cercidiphyllaceae and Daphniphyllaceae [23,24,26,84].These morphological affinities justify the close relationship between Altingiaceae and Hamamelidaceae.In addition, Altingiaceae, Cercidiphyllaceae, and Hamamelidaceae have stipules and winged seeds, distinguishing them from the nonstipulate leaves and wingless seeds of Daphniphyllaceae [23,24,26,84], supporting the transitional position of Cercidiphyllaceae as a phylogenetic link between Daphniphyllaceae and the clade Altingiaceae + Hamamelidaceae.The high levels of consistency between the nuclear genome phylogeny and morphological traits suggest that the cytonuclear discordance observed in the WCS phylogeny might have been caused by ancient interfamilial hybridization.
Interestingly, a previous study [56] showed that interfamilial hybridization might have occurred during the early evolution of the WCS.Based on scanning electron microscopic investigations, tricolpate-reticulate pollen was found to attach to the stigmas of the fossil pistillate inflorescences of Microaltingia, an extinct Altingiaceae genus from the late Cretaceous of the United States [56,85].Within the WCS, tricolpate-reticulate pollen exists in Hamamelidaceae, Cercidiphyllaceae, and Daphniphyllaceae but not in Altingiaceae [56,85].The fossil evidence implies that ancient hybridization between Altingiaceae and closely related families might be feasible.Therefore, chloroplast capture [17,82] caused by ancient hybridization is likely a reasonable interpretation of the phylogenetic discordances detected in the deep relationships within the WCS.
Concordant with fossil evidence [56,57,59,60,62,69,86], which suggests that the early evolution of the WCS might have experienced radiative lineage diversification, the results of divergence time estimation indicated that the WCS underwent rapid divergence in the crown groups during the early Cretaceous, leading to the occurrence of the stem lineage ancestors of Altingiaceae, Cercidiphyllaceae, Daphniphyllaceae, and Hamamelidaceae within a very short time span (within ∼4.56 Ma, and between 104.79 and 100.23 Ma).The mutually supporting evidence suggests that, in addition to ancient hybridization, incomplete lineage sorting (the stochastic sorting of ancestral sequence polymorphisms) [16,51] resulted from the process of radiative diversification during the early evolution of WSC may contribute to the observed phylogenetic recalcitrance in the deep relationships of the WCS.

Conclusion
The WCS is a representative of the phylogenetically recalcitrant node in the angiosperm tree of life, within which the deep relationships remain poorly resolved.Based on a broad taxonomic sampling at the genus level across the four currently recognized families in the WCS, we recovered a robust maternal backbone phylogeny for this group.Based on molecular and fossil evidence, this study indicates that the early evolution of the WCS might have undergone radiative diversification of crown groups in the early Cretaceous, ancient hybridization, and prominent extinction events during the transition between the Cretaceous and Paleocene.These events most likely resulted in the phylogenetic reconstruction of the deep relationships within the WCS being adversely affected by incomplete lineage sorting, cytonuclear discordance, and long-branch abstraction, which inevitably led to serious topological estimation biases.Nevertheless, possible ancient hybridization and incomplete lineage sorting in the early evolution of the WCS inferred in this study are merely based on the phylogenetic incongruence between plastid and nuclear genomic data, and other supporting evidence is still lacking.This inference needs to be validated through in-depth analysis of the conflicts between the species tree and gene trees using nuclear genome data containing multiple single-copy orthologous genes.
The genomic DNA of these newly sequenced samples was extracted from approximately 100 mg of silica geldried leaf tissue using a modified CTAB method [87].Then, the shotgun DNA libraries were constructed using a TruSeq DNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA) following the manufacturer's instructions.Paired-end sequencing was performed on an Illumina NovaSeq 6000 platform to generate approximately 4 Gbp of raw reads for each sample.

Plastome assembly and annotation
Trimmomatic v0.40 [88] was used to filter low-quality Illumina raw reads with pre-set parameters.The pipeline GetOrganelle v1.7.7.0 [89] was employed to assemble plastomes using Illumina clean reads with default parameters, using the plastomes of the corresponding species as a reference (Table S2).The assembled plastomes were further adjusted using Bandage v8.0 [90] and annotated using the online program GeSeq [91].Information on the initiation codon, stop codon, and intron sites of the protein-coding genes was examined and manually adjusted using Geneious v10.2 [92].tRNA genes were annotated using trnascan-SE v2.0 [93].The inverted repeat (IRa and IRb) regions of the plastome were determined using Geneious v10.2 [92].All WCS plastomes were progressively aligned with the complete plastome of Hamamelis mollis, as a reference, using the multiple genome alignment tool Mauve v4.0 [94], after one of the inverted repeat regions was removed.

Phylogenetic analysis
Given the close relationship between the WCS and Paeoniaceae [e.g., 27, 52], eight Paeonia species were selected as outgroups.In total, 78 plastid PCGs (Table S4) commonly shared by the sampled plastomes were extracted from each plastome for phylogenetic analysis using Phy-loSuite v1.2.2 [95].The PCGs were aligned with MAFFT v7.402 [96] and concatenated using Geneious v10.2 [92], with the default parameters.BI, ML, and MP methods were used to infer phylogenetic relationships.For BI analysis, PartitionFinder v2.1.1 [97] was used to estimate the best partitioning schemes and substitution models (Table S5).MrBayes v3.22 [98] based on BI, was used to construct a phylogenetic tree.The Markov Chain Monte Carlo (MCMC) analyses were run for two million generations, sampling one tree every 100 generations and discarding the first 25% of trees as burn-in.The obtained trees were evaluated for convergence using Tracer v1.7.1 [99], with effective sample sizes (ESSs) > 200.For the ML analysis, IQ-tree v2.1.3[100,101] was used to estimate the best partitioning schemes and substitution models (Table S6), and the ML bootstrap support (MLBS) value of each branch was calculated with 1000 replicates.The MP analyses employed PAUP* 4.a168 on the XSEDE platform via the Cyberinfrastructure for Phylogenetic Research Science (CIPRES) Gateway web server.All characters were treated as unordered and equally weighted, while branches with a minimum optimized length of zero were condensed [102].The analysis utilized a heuristic search approach with tree bisection-reconnection (TBR) branch swapping.It generated 1000 replicates, employing random-additionsequence methodology and allowing for the storage of up to 10,000 trees per replicate.From the retained mostparsimonious trees (MPTs), a strict consensus tree was derived.Bootstrap support values were calculated using bootstrap analyses [103] involving 1000 replicates.Each replicate consisted of 10 random-addition-sequence replicates, with a maximum of 100 trees saved per replicate.

Fig. 1
Fig. 1 Phylogenetic relationships within the "woody clade" in Saxifragales.The tree was built using maximum likelihood (ML) and Bayesian inference (BI) methods, based on 78 plastid protein-coding genes from 64 different species.(A) Cladogram.(B) Phylogram based on ML. (C) Phylogram based on BI.Numbers superimposed on the branches indicate bootstrap support (%) and posterior probability.FO, Fothergilleae; HA, Hamamelideae; EU, Eustigmateae; CO, Corylopsideae; LO, Loropetaleae deep clades, considering the ancient origin of their stem lineage ancestors (104.79-100.23 Ma) versus the relatively hysteretic divergence of their crown groups (37.81-4.61Ma).Within Hamamelidaceae, a similar pattern of early stem ages (100.3-83.07Ma) compared to relatively later crown ages (55.03-33.16Ma) was observed in the subfamilies Exbucklandioideae, Mytilarioideae, and Disanthoideae, as well as in the five tribes (Corylopsideae, Eustigmateae, Fothergilleae, Hamamelideae, and Loropetaleae) belonging to the subfamily Hamamelidoideae.As suggested in previous studies[8,16,21,27,29], the presence of deep stems within these clades may be attributed to a lack of cladogenesis or extensive extinction of closely related taxa during their early evolutionary processes.Fossil evidence has disproven the hypothesis that cladogenesis was absent during the early evolution of the aforementioned WCS clades.The most typical

Fig. 4
Fig. 4 Comparison of deep relationships within the "woody clade" in Saxifragales.(A) Phylogenetic analyses of plastomes.(B) Phylogenetic analyses of target-capture sequencing and transcriptome data